Compositions and methods for treatment of sepsis-related disorders

ABSTRACT

A method comprising administering, to a subject in need thereof, an effective amount of a nucleotide effective to disrupt one or more pathways leading to sepsis. The nucleotide may be a nitric oxide disruptor effective to decrease the expression of inducible nitric oxide synthase. The nitric oxide disrupter may comprise a polynucleotide strand exhibiting at least 70% sequence identity to one of Sequence ID No. 1 through Sequence ID No. 47. Additionally or alternatively, the nucleotide may be an α disintegrin and metalloproteinase (ADAM) enzyme inhibitor effective to decrease the expression of ADAM enzyme. The ADAM enzyme inhibitor may comprise a polynucleotide strand exhibiting at least 70% sequence identity to one of Sequence ID No. 48 through Sequence ID No. 56.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a filing under 35 U.S.C. 371 of InternationalApplication No. PCT/US2018/052050 filed Sep. 20, 2018, which iscontinuation of and claims priority to U.S. Provisional Application Ser.No. 62/562,060, filed Sep. 22, 2017, and entitled “Compositions andMethods for Treatment of Sepsis-Related Disorders” which is incorporatedherein by reference in its entirety.

REFERENCE TO SEQUENCE LISTING

The content of the ASCII text file of the sequence listing named“Sequence_Listing_ST25.txt”, which was filed in PCT/US2018/052050 onSep. 20, 2018, downloaded from the WIPO database, is 8.33 KB in size,was created on Sep. 20, 2018, and electronically submitted herewith thisapplication via the USPTO's EFS-Web patent application and documentsubmission system, is incorporated herein by reference in its entirety.

TECHNICAL FIELD

The present disclosure relates to compositions and methods for treatmentof sepsis and sepsis-related disorders. More particularly, the presentdisclosure relates to compositions and methods for the inhibition ofnitric oxide production by inducible nitric oxide synthase and/or theinhibition of an α disintegrin and metalloproteinase (ADAM) enzyme.

BACKGROUND

The currently accepted immunologic paradigm of sepsis suggests that thisdisorder is present when the activation of the systemic inflammatorypathways are triggered by infection. The infection initiates animmunologic response (inflammatory cytokine and eicosanoid/coagulationcascade) that propagates independently of the underlying infectioustrigger. Septic shock (sepsis with hypotension) has a mortality rate of40-60%, despite treatment.

Septic patients are in a hyperdynamic state characterized bytachycardia, high cardiac output, low systemic vascular resistance,hypoxemia, oliguria, and lactic acidosis. Following infection there isan increase in the circulating concentrations of catecholamines,cortisol, and glucagon resulting in tachycardia and peripheralvasoconstriction. This phase is followed by a progressive vasodilationassociated with high cardiac output and decreased vascular resistanceand, in some cases, vasoplegia. Subsequent to this, cardiac failuredevelops with a progressive fall in cardiac output and markeddisturbances in tissue perfusion and oxygenation occur. Hyperglycemia iscommonly present and represents the influence of circulating cytokinesand glucoregulatory hormones; however, in advanced stages hypoglycemiamay occur due to depletion of hepatic glycogen and inhibition ofgluconeogenesis. There are also associated marked increases in plasmatransaminase activity, urea and bilirubin concentrations, indicative ofhepatic and renal injury, while the development of lactic acidosisreflects inadequate tissue perfusion and anaerobic metabolism.

A principal factor in septic shock is the production of the signalingmolecule nitric oxide. Nitric oxide (NO), is produced by NO synthase(NOS) which utilizes 1-arginine and molecular oxygen as substrates andrequire the cofactors reduced nicotinamide-adenine-dinucleotidephosphate (NADPH), flavin adenine dinucleotide (FAD), flavinmononucleotide (FMN), and (6R-)5,6,7,8-tetrahydrobiopterin (BH(4)).Inducible NOS (“iNOS,” EC 1.14.13.39, one of three NOS isoforms)generates large amounts of NO that exert cytostatic effects.Specifically, the detrimental fall in blood pressure is predominantlyattributable to excess NO production by iNOS induced in the vascularwall.

Recently it has been shown that when short (18-30 bp) RNA duplexes areintroduced into mammalian cells in culture, sequence-specific inhibitionof target mRNA can be realized without inducing an interferon response.Certain of these short dsRNAs, referred to as small inhibitory RNAs(“siRNAs”), can act catalytically at sub-molar concentrations to cleavegreater than 95% of the target mRNA in the cell. These properties makesiRNAs potentially useful as therapeutic agents against diseases thatare caused by over-expression or mal-expression of genes such as theoverexpression of detrimental genes and gene products as a result ofsepsis or sepsis-related disorders.

SUMMARY

In some aspects disclosed herein is a method comprising administering,to a subject in need thereof, an effective amount of a nucleotideeffective to disrupt one or more pathways leading to sepsis. Thenucleotide may be a nitric oxide disruptor effective to decrease theexpression of inducible nitric oxide synthase by at least 50%, or by atleast 75%. The nitric oxide disrupter may comprise a polynucleotidestrand exhibiting at least 70% sequence identity to one of Sequence IDNo. 1 through Sequence ID No. 47, or exhibiting at least 80% sequenceidentity to one of Sequence ID No. 1 through Sequence ID No. 47, orexhibiting at least 90% sequence identity to one of Sequence ID No. 1through Sequence ID No. 47, or exhibiting at least 95% sequence identityto one of Sequence ID No. 1 through Sequence ID No. 47. Additionally oralternatively, the nucleotide may be an α disintegrin andmetalloproteinase (ADAM) enzyme inhibitor effective to decrease theexpression of ADAM enzyme by at least 50%, or by at least 75%. The ADAMenzyme inhibitor may comprise a polynucleotide strand exhibiting atleast 70% sequence identity to one of Sequence ID No. 48 throughSequence ID No. 56, or exhibiting at least 80% sequence identity to oneof Sequence ID No. 48 through Sequence ID No. 56, or exhibiting at least90% sequence identity to one of Sequence ID No. 48 through Sequence IDNo. 56 or exhibiting at least 95% sequence identity to one of SequenceID No. 48 through Sequence ID No. 56. The subject may be characterizedas exhibiting sepsis or a sepsis-related disorder.

In some aspects disclosed herein is a composition comprising apolynucleotide strand exhibiting at least 70% sequence identity to anyof SEQ ID NO.1 through SEQ ID NO 56. The polynucleotide strand mayexhibit at least 80% sequence identity to any of SEQ ID NO.1 through SEQID NO 47, or at least 90% sequence identity to any of SEQ ID NO.1through SEQ ID NO 47. Additionally or alternatively, the polynucleotidestrand exhibits at least 80% sequence identity to any of SEQ ID NO. 48through SEQ ID NO 56, or at least 90% sequence identity to any of SEQ IDNO. 48 through SEQ ID NO 56. The composition may be formulated fordelivery to a subject in need thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

For a more complete understanding of the present disclosure and theadvantages thereof, reference is now made to the following briefdescription, taken in connection with the accompanying drawings anddetailed description:

FIG. 1 is a diagram illustrating some of the interactions of variouscellular components leading to sepsis.

DETAILED DESCRIPTION

Disclosed herein are methods of treating a subject having sepsis or asepsis-related disorder comprising administering to the subject anoligonucleotide that disrupts one or more of the cellular pathwaysleading to sepsis (collectively, a “sepsis pathway disrupter”).

The term “subject” as used herein, refers to an animal which is theobject of treatment, observation, or experiment. By way of example only,a subject may be, but is not limited to, a mammal including, but notlimited to, a human. In some instances, the subject is a patient who isundergoing treatment for one or more medical conditions. The terms“treat,” “treating,” or “treatment,” as used herein, includealleviating, abating, or ameliorating a disease or condition, orsymptoms thereof; managing a disease or condition, or symptoms thereof;preventing additional symptoms; ameliorating or preventing theunderlying metabolic causes of symptoms; inhibiting the disease orcondition, e.g., arresting the development of the disease or condition;relieving the disease or condition; causing regression of the disease orcondition; relieving a symptom caused by the disease or condition;and/or stopping the symptoms of the disease or condition. Treatment, asused herein, also encompasses any pharmaceutical or medicinal use of thecompositions herein.

In an aspect, the subject is administered the compositions disclosedherein in a therapeutically effective amount sufficient for treating,preventing, and/or ameliorating one or more symptoms of a medicalcondition, disorder, disease, or dysfunction. Hereinafter, forsimplicity, the unwanted condition which has been used interchangeablywith the terms medical condition, disorder, disease, and dysfunction arecollectively referred to as the “medical condition.” As used herein,amelioration of the symptoms of the medical condition by administrationof a particular composition of the type disclosed herein refers to anylessening, whether lasting or transient, which can be attributed to orassociated with administration of compositions of the type disclosedherein. As used herein, a “therapeutically effective amount” means asufficient amount of the compositions disclosed herein to treat,prevent, and/or ameliorate one or more symptoms of the medicalcondition. It also may include a safe and tolerable amount of thecompositions disclosed herein, as based on industry and/or regulatorystandards. As will be understood by the ordinarily skilled artisan, anamount that proves to be a “therapeutically effective amount” in a giveninstance, for a particular subject, may not be effective for 100% ofsubjects similarly treated for the medical condition underconsideration, even though such dosage is deemed a “therapeuticallyeffective amount” by ordinarily skilled practitioners. Thetherapeutically effective amount for a particular individual may varydepending on numerous factors such as the nature of the medicalcondition, severity of the medical condition, subject weight, subjectage, and the general health of the subject. It is contemplated that thetherapeutically effective amount may be optimized by one or morehealthcare professionals in consideration of the particular factorsaffecting a subject.

The phrase “gene silencing” refers to a process by which the expressionof a specific gene product is lessened or attenuated. Gene silencing cantake place by a variety of pathways. Unless specified otherwise, as usedherein, gene silencing refers to decreases in gene product expressionthat results from RNA interference (RNAi), a defined, though partiallycharacterized pathway whereby small inhibitory RNA (siRNA) act inconcert with host proteins (e.g., the RNA induced silencing complex,RISC) to degrade messenger RNA (mRNA) in a sequence-dependent fashion.The level of gene silencing can be measured by a variety of means,including, but not limited to, measurement of transcript levels byNorthern Blot Analysis, B-DNA techniques, transcription-sensitivereporter constructs, expression profiling (e.g., DNA chips), and relatedtechnologies. Alternatively, the level of silencing can be measured byassessing the level of the protein encoded by a specific gene. This canbe accomplished by performing a number of studies including WesternAnalysis, measuring the levels of expression of a reporter protein thathas e.g., fluorescent properties (e.g., GFP) or enzymatic activity(e.g., alkaline phosphatases), or several other procedures.

In an aspect, the method of treatment comprises administering acomposition comprising oligonucleotide that may disrupt the productionof iNOS. For example, FIG. 1 illustrates a portion of the interactionsof various cellular components potentially leading to sepsis. As shownin FIG. 1 , administration of a composition effective to disrupt theproduction of iNOS 100 may disrupt one or more of the cellular pathwaysleading to sepsis.

In an aspect, gene silencing of iNOS using any of the methodologies orcompositions disclosed herein results in a reduction in the circulatingconcentration of nitric oxide. For example, in some aspects, the sepsispathway disrupter is a nitric oxide disruptors (NOD).

In an aspect, the compositions disclosed herein result in substantialsilencing of the iNOS gene and consequently the reduced production ofnitric oxide. As used herein the term “substantial silencing” means thatthe mRNA of the targeted allele is inhibited and/or degraded by thepresence of the introduced NOD, such that expression of the targetedallele is reduced by about 10% to 100% as compared to the level ofexpression seen when the NOD is not present. Generally, when an alleleis substantially silenced, it will have at least 40%, 50%, 60%, to 70%,e.g., 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, to 79%, generally at least80%, e.g., 81%-84%, at least 85%, e.g., 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or even 100% reduction expressionas compared to when the NOD is not present. As used herein the term“substantially normal activity” means the level of expression of anallele when a NOD has not been introduced. The NODs disclosed hereincomprise nucleic acids or nucleotides. The term “nucleotide” refers to aribonucleotide or a deoxyribonucleotide or modified form thereof, aswell as an analog thereof. Nucleotides include species that comprisepurines, e.g., adenine, hypoxanthine, guanine, and their derivatives andanalogs, as well as pyrimidines, e.g., cytosine, uracil, thymine, andtheir derivatives and analogs. Nucleotide analogs include nucleotideshaving modifications in the chemical structure of the base, sugar and/orphosphate, including, but not limited to, 5-position pyrimidinemodifications, 8-position purine modifications, modifications atcytosine exocyclic amines, and substitution of 5-bromo-uracil; and2′-position sugar modifications, including but not limited to,sugar-modified ribonucleotides in which the 2′-OH is replaced by a groupsuch as an H, OR, R, halo, SH, SR, NH₂, NHR, NR₂, or CN, wherein R is analkyl moiety. Nucleotide analogs are also meant to include nucleotideswith bases such as inosine, queuosine, xanthine, sugars such as2′-methyl ribose, non-natural phosphodiester linkages such asmethylphosphonates, phosphorothioates and peptides. As used herein, theterm “nucleic acid” or “nucleic acid molecule” refers topolynucleotides, such as deoxyribonucleic acid (DNA) or ribonucleic acid(RNA), oligonucleotides, fragments generated by the polymerase chainreaction (PCR), and fragments generated by any of ligation, scission,endonuclease action, and exonuclease action. Nucleic acid molecules canbe composed of monomers that are naturally-occurring nucleotides (suchas DNA and RNA), or analogs of naturally-occurring nucleotides (e.g.,.alpha.-enantiomeric forms of naturally-occurring nucleotides), or acombination of both. Modified nucleotides can have alterations in sugarmoieties and/or in pyrimidine or purine base moieties. Sugarmodifications include, for example, replacement of one or more hydroxylgroups with halogens, alkyl groups, amines, and azido groups, or sugarscan be functionalized as ethers or esters. Moreover, the entire sugarmoiety can be replaced with sterically and electronically similarstructures, such as aza-sugars and carbocyclic sugar analogs. Examplesof modifications in a base moiety include alkylated purines andpyrimidines, acylated purines or pyrimidines, or other well-knownheterocyclic substitutes. Nucleic acid monomers can be linked byphosphodiester bonds or analogs of such linkages. Analogs ofphosphodiester linkages include phosphorothioate, phosphorodithioate,phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate,phosphoranilidate, phosphoramidate, and the like. The term “nucleic acidmolecule” also includes so-called “peptide nucleic acids,” whichcomprise naturally-occurring or modified nucleic acid bases attached toa polyamide backbone. Nucleic acids can be either single stranded ordouble stranded.

In an aspect, the NOD is a small interfering RNA (siRNA). Naturallyoccurring RNAi, a double-stranded RNA (dsRNA) is cleaved by an RNaseIII/helicase protein, Dicer, into small interfering RNA (siRNA)molecules, a dsRNA of 19-27 nucleotides (nt) with 2-nt overhangs at the3′ ends. siRNAs are incorporated into a multicomponent-ribonucleasecalled RNA-induced silencing complex (RISC). One strand of siRNA remainsassociated with RISC and guides the complex toward a cognate RNA thathas sequence complementary to the guider ss-siRNA in RISC. ThissiRNA-directed endonuclease digests the RNA, thereby inactivating it.

As identified in the SEQUENCE LISTING which forms a part of thisdisclosure, Sequence ID No. 1 through Sequence ID No. 47 (i.e., <210>1through <210>47) are representative of the sense strand of the NODsdescribed herein. Consequently, the present disclosure contemplates theuse of siRNA comprising the sense strands of any of Sequence ID No. 1through Sequence ID No. 47 and its complementary strand. In an aspect,the siRNAs of the present disclosure comprise the polynucleotides of anyof Sequence ID No 1 through Sequence ID No 47 and its perfectcomplement, alternatively the polynucleotides of any of Sequence ID No 1through Sequence ID No 47 and a complementary strand. The term“complementary” refers to the ability of polynucleotides to form basepairs with one another. Base pairs are typically formed by hydrogenbonds between nucleotide units in antiparallel polynucleotide strands.

Complementary polynucleotide strands can base pair in the Watson-Crickmanner (e.g, A to T, A to U, C to G), or in any other manner that allowsfor the formation of duplexes. As persons skilled in the art are aware,when using RNA as opposed to DNA, uracil rather than thymine is the basethat is considered to be complementary to adenosine. However, when a Uis denoted in the context of the present disclosure, the ability tosubstitute a T is implied, unless otherwise stated.

Perfect complementarity or 100% complementarity refers to the situationin which each nucleotide unit of one polynucleotide strand can hydrogenbond with a nucleotide unit of a second polynucleotide strand. Less thanperfect complementarity refers to the situation in which some, but notall, nucleotide units of two strands can hydrogen bond with each other.For example, for two 20-mers, if only two base pairs on each strand canhydrogen bond with each other, the polynucleotide strands exhibit 10%complementarity. In the same example, if 18 base pairs on each strandcan hydrogen bond with each other, the polynucleotide strands exhibit90% complementarity

In an aspect, a NOD comprises a siRNA, a functional variant thereof; orcombinations thereof. In some aspects, a functional variant of an siRNAdisclosed herein comprises at least 70% sequence identity with anysequence disclosed herein, alternatively at least 75%, alternatively atleast 80%, alternatively at least 85%, alternatively at least 90% oralternatively at least 95%.

In general, “identity” refers to an exact nucleotide-to-nucleotidecorrespondence of two oligonucleotides or polynucleotides sequences.Percent identity can be determined by a direct comparison of thesequence information between two molecules by aligning the sequences,counting the exact number of matches between the two aligned sequences,dividing by the length of the shorter sequence, and multiplying theresult by 100. Readily available computer programs can be used to aid inthe analysis, such as Wisconsin Sequence Analysis Package, Version 8(available from Genetics Computer Group, Madison, Wis.) for example, theBESTFIT, FASTA and GAP programs, which rely on the Smith and Watermanalgorithm. These programs are readily utilized with the defaultparameters recommended by the manufacturer and described in theWisconsin Sequence Analysis Package referred to above. For example,percent identity of a particular nucleotide sequence to a referencesequence can be determined using the homology algorithm of Smith andWaterman with a default scoring table and a gap penalty of sixnucleotide positions.

Alternatively, homology can be determined by hybridization ofpolynucleotides under conditions which form stable duplexes betweenhomologous regions, followed by digestion with single-stranded-specificnuclease(s), and size determination of the digested fragments. DNAsequences that are substantially homologous can be identified in aSouthern hybridization experiment under, for example, stringentconditions, as defined for that particular system. Appropriatehybridization conditions may be defined using any suitable methodology.

In some aspects, one or more of the nucleotides present in the siRNA maybe modified to achieve one or more user and/or process goals, such asincreased stability. Modified bases refer to nucleotide bases such as,for example, adenine, guanine, cytosine, thymine, uracil, xanthine,inosine, and queuosine that have been modified by the replacement oraddition of one or more atoms or groups. Some examples of types ofmodifications that can comprise nucleotides that are modified withrespect to the base moieties include but are not limited to, alkylated,halogenated, thiolated, aminated, amidated, or acetylated bases,individually or in combination. More specific examples include, forexample, 5-propynyluridine, 5-propynylcytidine, 6-methyladenine,6-methylguanine, N,N,-dimethyladenine, 2-propyladenine, 2-propylguanine,2-aminoadenine, 1-methylinosine, 3-methyluridine, 5-methylcytidine,5-methyluridine and other nucleotides having a modification at the 5position, 5-(2-amino)propyl uridine, 5-halocytidine, 5-halouridine,4-acetylcytidine, 1-methyladenosine, 2-methyladenosine,3-methylcytidine, 6-methyluridine, 2-methylguanosine, 7-methylguanosine,2,2-dimethylguanosine, 5-methylaminoethyluridine, 5-methyloxyuridine,deazanucleotides such as 7-deaza-adenosine, 6-azouridine, 6-azocytidine,6-azothymidine, 5-methyl-2-thiouridine, other thio bases such as2-thiouridine and 4-thiouridine and 2-thiocytidine, dihydrouridine,pseudouridine, queuosine, archaeosine, naphthyl and substituted naphthylgroups, any 0- and N-alkylated purines and pyrimidines such asN6-methyladenosine, 5-methylcarbonylmethyluridine, uridine 5-oxyaceticacid, pyridine-4-one, pyridine-2-one, phenyl and modified phenyl groupssuch as aminophenol or 2,4,6-trimethoxy benzene, modified cytosines thatact as G-clamp nucleotides, 8-substituted adenines and guanines,5-substituted uracils and thymines, azapyrimidines, carboxyhydroxyalkylnucleotides, carboxyalkylaminoalkyl nucleotides, andalkylcarbonylalkylated nucleotides. Modified nucleotides also includethose nucleotides that are modified with respect to the sugar moiety, aswell as nucleotides having sugars or analogs thereof that are notribosyl. For example, the sugar moieties may be, or be based on,mannoses, arabinoses, glucopyranoses, galactopyranoses, 4′-thioribose,and other sugars, heterocycles, or carbocycles. The term nucleotide isalso meant to include what are known in the art as universal bases. Byway of example, universal bases include but are not limited to3-nitropyrrole, 5-nitromdole, or nebularine. The term “nucleotide” isalso meant to include the N3′ to P5′ phosphoramidate, resulting from thesubstitution of a ribosyl 3′ oxygen with an amine group. Further, theterm nucleotide also includes those species that have a detectablelabel, such as for example a radioactive or fluorescent moiety, or masslabel attached to the nucleotide.

Without wishing to be limited by theory, utilization of a NOD of thetype disclosed herein would diminish efficient transcription of iNOSmRNA, reduce successful movement of guide strand mRNA to translation andinterfere with efficient translation of mRNA which produces iNOS andsubsequently nitric oxide.

Additionally or alternatively, in an aspect the method of treatmentcomprises administering a composition comprising oligonucleotide thatmay inhibit an α disintegrin and metalloproteinase (ADAM) enzyme,particularly, ADAM 17 (synonyms: CD156b; cSVP; MGC71942; TACE). Forexample, in some aspects, the sepsis pathway disrupter is an inhibitorsof ADAM 17, hereinafter termed A17 inhibitors (“A17I”). A17I arematerials which result in a down-regulation or reduction in the activityof ADAM 17. ADAM 17 is a member of the ADAM family of enzymes which areZn²⁺-dependent, modular cell surface proteins belonging to theadamalysin protein family. They are closely related to othermetalloenzymes such as ADAM-TSs (ADAMs with thrombospondin domains),matrix metalloproteinases (MMPs) and snake venom metalloproteinases(SVMP). The structure of ADAMs closely resembles the Class III snakevenom metalloenzymes or reprolysines and it forms the basis of the manyADAM enzyme functions. ADAM-17 was discovered in 1997 and describedsimultaneously by two research groups as the enzyme that releasesmembrane bound tumor necrosis factor (TNF)-α precursor to a solubleform. This discovery was significant because TNFα is critical ininflammatory processes. ADAM-17 was described as a protein of 824 aminoacids (accession number NM_003183), and its gene is located onchromosome 2p25. ADAM-17 is widely expressed in various tissuesincluding the brain, heart, kidney, and skeletal muscle and itsexpression changes during embryonic development and adult life. ADAM-17is a multi-domain protein starting with a signal sequence (1-17 aa),followed by a prodomain (18-214 aa), a metalloenzyme or catalytic domain(215-473 aa) with the typical HEXXHXXGXXH (X being any amino acidresidue) sequence, α disintegrin domain (474-572 aa), an cysteine-richdomain (603-671 aa), followed by a transmembrane domain (672-694 aa) anda cytoplasmic tail (695-824 aa). The most well-known function ofADAM-17, is to cleave ectodomains of various transmembrane proteins.Proteins with different functions can be processed by ectodomainshedding: EGFR ligands, proinflammatory cytokines like TNFα and itsreceptor TNFRI, adhesion molecules and the amyloid precursor protein.

The extent of downregulation of ADAM17 or its gene product may bedetermined using any suitable assay. Suitable assays include withoutlimitation, e.g., examination of protein or mRNA levels using anysuitable technique such as dot blots, northern blots, in situhybridization, ELISA, microarray hybridization, immunoprecipitation,enzyme function, as well as phenotypic assays known to those of skill inthe art. To examine the extent of gene silencing, a test sample (e.g., abiological sample from organism of interest expressing the targetgene(s) or a sample of cells in culture expressing the target gene(s))is contacted with a A17I that silences, reduces, or inhibits expressionof the target gene(s). Expression of the target gene in the test sampleis compared to expression of the target gene in a control sample (e.g.,a biological sample from organism of interest expressing the target geneor a sample of cells in culture expressing the target gene) that is notcontacted with the A17I. Control samples (i.e., samples expressing thetarget gene) are assigned a value of 100%. In an aspect, substantialsilencing, inhibition, down-regulation or reduction of expression of atarget gene is achieved when the value of test the test sample relativeto the control sample is about 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%,55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, or 10%.

In an aspect the A17I is a microRNA (miRNA, miR). miRs refer tosingle-stranded RNA molecules that are generally 21-23 nucleotides inlength which regulate gene expression. MicroRNAs are processed fromprimary transcripts known as pri-miRNA to short stem-loop structurescalled precursor (pre)-miRNA and finally to functional, mature microRNA.Mature microRNA molecules are partially complementary to one or moremessenger RNA molecules, and their primary function is to down-regulategene expression through the RNAi pathway.

In an aspect, the A17I is a small interfering RNA (siRNA). Naturallyoccurring RNAi, a double-stranded RNA (dsRNA) is cleaved by an RNaseIII/helicase protein, Dicer, into small interfering RNA (siRNA)molecules, a dsRNA of 19-27 nucleotides (nt) with 2-nt overhangs at the3′ ends. The siRNAs may be incorporated into amulticomponent-ribonuclease called RNA-induced silencing complex (RISC).One strand of siRNA remains associated with RISC and guides the complextoward a cognate RNA that has sequence complementary to the guiderss-siRNA in RISC. This siRNA-directed endonuclease digests the RNA,thereby inactivating it. These and other characteristics of RISC, siRNAmolecules, and RNAi have been described.

In an aspect, the A17I is an antisense oligonucleotide. Antisenseoligonucleotides (ASOs) are synthetic nucleic acids that bind to acomplementary target and suppress function of that target. Typically,ASOs are used to reduce or alter expression of RNA targets, particularlymessenger RNA (mRNA) or microRNA (miRNA) species. As a generalprinciple, ASOs can suppress gene expression via two differentmechanisms of action, including: 1) by steric blocking, wherein the ASOtightly binds the target nucleic acid and inactivates that species,preventing its participation in cellular biology, or 2) by triggeringdegradation, wherein the ASO binds the target and leads to activation ofa cellular nuclease that degrades the targeted nucleic acid species. Oneclass of “target degrading” ASOs are “RNase H active”; formation ofheteroduplex nucleic acids by hybridization of the target RNA with aDNA-containing “RNase H active” ASO forms a substrate for the enzymeRNase H. RNase H degrades the RNA portion of the heteroduplex molecule,thereby reducing expression of that species. Degradation of the targetRNA releases the ASO, which is not degraded, which is then free torecycle and can bind another RNA target of the same sequence.

In an aspect, an A17I comprises a microRNA, a siRNA, an ASO, an iRNA, aniRNA agent, a shRNA, a functional variant thereof; or combinationsthereof. In some aspects, a functional variant of an oligonucleotidedisclosed herein comprises at least 70% sequence identity with anysequence disclosed herein, or at least 75%, or at least 80%, or at least85%, or at least 90%, or at least 95% sequence identity. In general,“identity” refers to an exact nucleotide-to-nucleotide correspondence oftwo oligonucleotides or polynucleotides sequences. Percent identity canbe determined by a direct comparison of the sequence information betweentwo molecules by aligning the sequences, counting the exact number ofmatches between the two aligned sequences, dividing by the length of theshorter sequence, and multiplying the result by 100. Readily availablecomputer programs can be used to aid in the analysis, such as WisconsinSequence Analysis Package, Version 8 (available from Genetics ComputerGroup, Madison, Wis.) for example, the BESTFIT, FASTA and GAP programs,which rely on the Smith and Waterman algorithm. These programs arereadily utilized with the default parameters recommended by themanufacturer and described in the Wisconsin Sequence Analysis Packagereferred to above. For example, percent identity of a particularnucleotide sequence to a reference sequence can be determined using thehomology algorithm of Smith and Waterman with a default scoring tableand a gap penalty of six nucleotide positions.

Alternatively, homology can be determined by hybridization ofpolynucleotides under conditions which form stable duplexes betweenhomologous regions, followed by digestion with single-stranded-specificnuclease(s), and size determination of the digested fragments. DNAsequences that are substantially homologous can be identified in aSouthern hybridization experiment under, for example, stringentconditions, as defined for that particular system. Defining appropriatehybridization conditions is within the skill of the art.

As identified in the SEQUENCE LISTING which forms a part of thisdisclosure, Sequence ID No. 48 through Sequence ID No. 56 (i.e., <210>48through <210>56) are representative of the A17Is described herein. In anaspect, the A17I comprises an oligonucleotide having any one of SequenceID No. 48 through Sequence ID No. 56, alternatively a functional variantthereof. In some aspects, a A17I suitable for use in the presentdisclosure comprises at least 70% sequence identity with any sequencedisclosed herein, alternatively at least 75%, alternatively at least80%, alternatively at least 85%, alternatively at least 90% oralternatively at least 95%.

In an aspect, the A17I has from about 20% to about a 90% modification orfrom about a 40% to about 60% modification.

In an aspect, the sepsis pathway disrupters of this disclosure may beincluded within or comprise a component of a formulation foradministration to a subject. The term “formulation” as used hereinrefers to its generally-accepted meaning in the art, for example, to acomposition such as in a pharmaceutically acceptable carrier or diluent,in a form suitable for administration, for example, systemic or localadministration, into a cell or subject such as a human. Suitable forms,in part, depend upon the use or the route of entry, for example oral,transdermal, inhalation, or by injection. Such forms should not preventthe formulation from reaching a target cell (i.e., a cell to which thenucleic acid is desirable for delivers). For example, compositionsinjected into the blood stream should be soluble. Other factors includeconsiderations such as toxicity and forms that prevent the compositionor formulation from exerting its effect.

In an aspect, a sepsis pathway disrupter may be formulated foradministration to a subject suffering from sepsis or a sepsis-relateddisorder. The choice of formulation and delivery route for a sepsispathway disrupter may be driven by patient acceptability, the propertiesof the sepsis pathway disrupter (such as its solubility), access to adisease location, or effectiveness in dealing with the specific disease.

In an aspect, the sepsis pathway disrupter may be formulated foradministration via a peroral route. For example, in an aspect, thesepsis pathway disrupter is delivered via injection for example,following mixing with saline.

In an aspect, the sepsis pathway disrupter may be formulated foradministration via pulmonary delivery. Pulmonary delivery may beeffected in a variety of ways—via aerosols, metered dose inhaler systems(MDIs), powders (dry powder inhalers, DPIs) and solutions (nebulizers),all of which may contain nanostructures such as liposomes, micelles,nanoparticles and dendrimers. Pulmonary delivery to be a viable optionfor the delivery of drugs systemically.

In an aspect, the sepsis pathway disrupter may be formulated foradministration transdermally. Transdermal delivery avoids problems suchas gastrointestinal irritation, metabolism, variations in delivery ratesand interference due to the presence of food. It is also suitable forunconscious patients. The technique is generally non-invasive andaesthetically acceptable, and can be used to provide local delivery overseveral days.

In an aspect, the sepsis pathway disrupter may be formulated foradministration parenterally. Trans-tissue and local delivery systems mayproduce an elevated pharmacological effect, while minimizing systemic,administration-associated toxicity. Trans-tissue delivery systemsinclude: drug-loaded gelatinous gels, which are formed in-situ andadhere to resected tissues, releasing drugs, proteins or gene-encodingadenoviruses; antibody-fixed gelatinous gels (cytokine barrier) thatform a barrier, which, on a target tissue could prevent the permeationof cytokines into that tissue; cell-based delivery, which involves agene-transduced oral mucosal epithelial cell (OMEC)-implanted sheet;device-directed delivery—a rechargeable drug infusion device that can beattached to the resected site.

Once the sepsis pathway disrupters are delivered intra-cellularly,affinity dynamics with the components of the nuclear, cytoplasmic andribosomal elements will determine their effectiveness in reducingproduction and decreasing the intensity of sepsis and sepsis-relateddisorders.

In an aspect, the sepsis pathway disrupter may be chemicallysynthesized. Oligonucleotides (e.g., certain modified oligonucleotidesor portions of oligonucleotides lacking ribonucleotides) may besynthesized using protocols known in the art, for example as describedin Caruthers et al., 1992, Methods in Enzymology 211, 3-19, Thompson etal., International PCT Publication No. WO 99/54459, Wincott et al.,1995, Nucleic Acids Res. 23, 2677-2684, Wincott et al., 1997, MethodsMol. Bio., 74, 59, Brennan et al., 1998, Biotechnol Bioeng. 61, 33-45,and Brennan, U.S. Pat. No. 6,001,311. The synthesis of oligonucleotidesmakes use of common nucleic acid protecting and coupling groups, such asdimethoxytrityl at the 5′-end, and phosphoramidites at the 3′-end.

Alternatively, a sepsis pathway disrupter may be expressed and deliveredvia a transcript inserted into DNA or RNA vectors. The recombinantvectors can be DNA plasmids or viral vectors. Non-limiting examples ofsepsis pathway disrupter-expressing viral vectors can be constructedbased on adeno-associated virus, retrovirus, adenovirus, or alphavirus.

In some aspects, pol III-based constructs are used to express the sepsispathway disrupter. Transcription of the siNA molecule sequences can bedriven from a promoter for eukaryotic RNA polymerase I (pol I), RNApolymerase II (pol II), or RNA polymerase III (pol III), (see forexample, Thompson, U.S. Pat. Nos. 5,902,880 and 6,146,886). Transcriptsfrom pol II or pol III promoters may be expressed at high levels; thelevels of a given pol II promoter in a given cell type depends on thenature of the gene regulatory sequences (enhancers, silencers, etc.)present nearby. Prokaryotic RNA polymerase promoters may also be used,provided that the prokaryotic RNA polymerase enzyme is expressed in theappropriate cells. These exemplary transcription units can beincorporated into a variety of vectors for introduction into mammaliancells, including but not restricted to, plasmid DNA vectors, viral DNAvectors (such as adenovirus or adeno-associated virus vectors). or viralRNA vectors (such as retroviral or alphavirus vectors)

Vectors used to express the sepsis pathway disrupter of the presentdisclosure can encode one or both strands of a siNA duplex, or a singleself-complementary strand that self hybridizes into an siRNA duplex. Thenucleic acid sequences encoding the sepsis pathway disrupter of thepresent disclosure can be operably linked in a manner that allowsexpression of the sepsis pathway disrupter. In some aspects, theconstructs comprising sepsis pathway disrupter may additionally comprisereporter genes (e.g., green fluorescent protein) and selection genes(e.g., for antibiotic resistance).

In one aspect, a carrier system may contain the sepsis pathwaydisrupter, for example, for delivery. A sepsis pathway disrupterassociated with one or more of the delivery vehicles disclosed hereinmay be referred to as a packaged sepsis pathway disrupter.” Non-limitingexamples of delivery vehicles suitable for formulation of the nucleicacid molecules of the instant present disclosure include: LipidNanoparticles (see for example Semple et al., 2010, Nat Biotechnol.,February; 28(2):172-6); P-glycoprotein inhibitors (such as PluronicP85); biodegradable polymers, such as poly (DL-lactide-coglycolide)microspheres for sustained release delivery (Emerich, D F et al, 1990,Cell Transplant, 8, 47-58); and loaded nanoparticles, such as those madeof polybutylcyanoacrylate. Other non-limiting examples of deliverystrategies for the nucleic acid molecules of the instant presentdisclosure include material described in Boado et al., 1998, J. Pharm.Sci., 87, 1308-1315; Tyler et al., 1999, FEBS Lett., 421, 280-284;Partridge et al., 1995, PNAS USA., 92, 5592-5596; Boado, 1995, Adv. DrugDelivery Rev., 15, 73-107; Aldrian-Herrada et al., 1998, Nucleic AcidsRes., 26, 4910-4916; and Tyler et al., 1999, PNAS USA., 96, 7053-7058. Apackaged sepsis pathway disrupter may be further modified to improveproperties such as bioavailiability by modification of the packaging(e.g.,) using any suitable methodology (e.g., conjugation with atargeting molecule).

In an aspect, a sepsis pathway disrupter may be a component of a kit andmay be formulated as a pharmaceutical composition. For example, thesepsis pathway disrupter may be associated with colloidal drug carriersystems such as micellar solutions, vesicle and liquid crystaldispersions, as well as nanoparticle dispersions consisting of smallparticles of 10-400 nm diameter.

In an aspect, a sepsis pathway disrupter may be associated with aliposome or niosome. Liposomes are a form of vesicles that consisteither of many, few or just one phospholipid bilayers. The polarcharacter of the liposomal core enables polar drug molecules to beencapsulated. Amphiphilic and lipophilic molecules are solubilizedwithin the phospholipid bilayer according to their affinity towards thephospholipids. Participation of nonionic surfactants instead ofphospholipids in the bilayer formation results in niosomes. siRNA of thetype disclosed herein (i.e., sepsis pathway disrupters) can beincorporated without loss of their activity within the hydrophobicdomain of vesicle membranes, acting as a size-selective filter, onlyallowing passive diffusion of small solutes such as ions, nutrients andantibiotics. Thus, sepsis pathway disrupters may be encapsulated in ananocage and are effectively protected from premature degradation byproteolytic enzymes.

In an aspect, a sepsis pathway disrupter may be associated with adendrimer. Dendrimers are nanometer-sized, highly branched andmonodisperse macromolecules with symmetrical architecture. They consistof a central core, branching units and terminal functional groups. Thecore together with the internal units, determine the environment of thenanocavities and consequently their solubilizing properties, whereas theexternal groups the solubility and chemical behavior of these polymers.Targeting effectiveness is affected by attaching targeting ligands atthe external surface of dendrimers, while their stability and protectionfrom the Mononuclear Phagocyte System (MPS) is being achieved byfunctionalization of the dendrimers with polyethylene glycol chains(PEG).

In an aspect, the sepsis pathway disrupter may be associated with aliquid crystal. Liquid crystals combine the properties of both liquidand solid states. They can be made to form different geometries, withalternative polar and non-polar layers (i.e., a lamellar phase) whereaqueous drug solutions can be included.

In an aspect, the sepsis pathway disrupter may be associated with ananoparticle. Nanoparticles (including nanospheres and nanocapsules ofsize 10-200 nm) are in the solid state and are either amorphous orcrystalline. They are able to adsorb and/or encapsulate a drug, thusprotecting it against chemical and enzymatic degradation. Nanocapsulesare vesicular systems in which the drug is confined to a cavitysurrounded by a unique polymer membrane, while nanospheres are matrixsystems in which the drug is physically and uniformly dispersed.Nanoparticles as drug carriers can be formed from both biodegradablepolymers and non-biodegradable polymers. In recent years, biodegradablepolymeric nanoparticles have attracted considerable attention aspotential delivery devices in view of their applications in thecontrolled release of drugs, in targeting particular organs/tissues, ascarriers of DNA in gene therapy, and in their ability to deliverproteins, peptides and genes through the peroral route.

In an aspect, the sepsis pathway disrupter may be associated with ahydrogel. Hydrogels are three-dimensional, hydrophilic, polymericnetworks capable of imbibing large amounts of water or biologicalfluids. The networks are composed of homopolymers or copolymers, and areinsoluble due to the presence of chemical crosslinks (tie-points,junctions), or physical crosslinks, such as entanglements orcrystallites. Hydrogels exhibit a thermodynamic compatibility withwater, which allows them to swell in aqueous media. They are used toregulate drug release in reservoir-based, controlled release systems oras carriers in swellable and swelling-controlled release devices. On theforefront of controlled delivery, hydrogels as enviro-intelligent andstimuli-sensitive gel systems modulate release in response to pH,temperature, ionic strength, electric field, or specific analyteconcentration differences. In these systems, release can be designed tooccur within specific areas of the body (e.g., within a certain pH ofthe digestive tract) or also via specific sites (adhesive orcell-receptor specific gels via tethered chains from the hydrogelsurface). Hydrogels as delivery systems can be very promising materialsif combined with the technique of molecular imprinting.

In an aspect, the sepsis pathway disrupter may be a component of aconjugate or complex provided that can impart therapeutic activity bytransferring therapeutic compounds across cellular membranes, alteringthe pharmacokinetics, and/or modulating the localization of nucleic acidmolecules of the present disclosure. For example, the conjugate cancomprise polyethylene glycol (PEG) can be covalently attached to asepsis pathway disrupter. The attached PEG can be any molecular weight,for example from about 100 to about 50,000 daltons (Da).

In an aspect, the sepsis pathway disrupter may be a component of acomposition or formulation comprising surface-modified liposomescontaining poly (ethylene glycol) lipids (PEG-modified, orlong-circulating liposomes, or stealth liposomes) and sepsis pathwaydisrupter. In some aspects, the sepsis pathway disrupter can also beformulated or complexed with polyethyleneimine and derivatives thereof,such as polyethyleneimine-polyethyleneglycol-N-acetylgalactosamine(PEI-PEG-GAL) orpolyethyleneimine-polyethyleneglycol-tri-N-acetylgalactosamine(PEI-PEG-triGAL) derivatives.

In an aspect, the formulation may contain additional ingredients. Asused herein, “additional ingredients” include, but are not limited to,one or more of the following: excipients; surface active agents;dispersing agents; inert diluents; granulating and disintegratingagents; binding agents; lubricating agents; sweetening agents; flavoringagents; coloring agents; preservatives; physiologically degradablecompositions such as gelatin; aqueous vehicles and solvents; oilyvehicles and solvents; suspending agents; dispersing or wetting agents;emulsifying agents, demulcents; buffers; salts; thickening agents;fillers; emulsifying agents; antioxidants; antibiotics; antifungalagents; stabilizing agents; and pharmaceutically acceptable polymeric orhydrophobic materials.

In an aspect, a method of treating a subject having sepsis or asepsis-related disorder comprises administering to a subject in needthereof a sepsis pathway disrupter of the type disclosed herein. Forexample, a subject in need thereof may present with sepsis or asepsis-related disorder. Clinical presentation of sepsis or asepsis-related disorder may be observed in subjects demonstratinghypotension with low systemic vascular resistance. A trend from normalhemodynamics to a septic state or who are evaluated as being at highrisk of clinical presenting with sepsis or a sepsis-related disorder maybe administered a continuous infusion with or without a loading or bolusdose of a sepsis pathway disrupter of the type disclosed herein.Administration of a sepsis pathway disrupter comprising a NOD asdisclosed herein may result in the reduction or arrest of the productionof new endothelial inducible nitric oxide synthase enzymes, resulting ina reduced production of nitric oxide and its resultant vasodilatoryeffects. Additionally or alternatively, administration of a sepsispathway disrupter comprising a A17I as disclosed herein may result in adown-regulation or reduction in the activity of ADAM 17. In variousaspects, a method of the present disclosure comprises administration ofan effective amount of a sepsis pathway disrupter of the type disclosedherein to a subject experiencing sepsis or a sepsis-related disorder mayadvantageously result in the subject experiencing a normalization ofsystemic vascular resistance with improved tissue perfusion andoxygenation, reduction of the need for endogenous and administeredcatecholamine infusions and diminished demands on cardiac output.

What is claimed is:
 1. A method comprising administering an effectiveamount of a nitric oxide disruptor to a subject in need thereof, whereinthe nitric oxide disruptor is a small interfering RNA and exhibits atleast 70% sequence identity to one of Sequence ID No. 1 through SequenceID No.
 25. 2. The method of claim 1, wherein the nitric oxide disrupteris effective to decrease the expression of inducible nitric oxidesynthase by at least 50%.
 3. The method of claim 1, wherein the nitricoxide disrupter is effective to decrease the expression of induciblenitric oxide synthase by at least 75%.
 4. The method of claim 2, whereinthe nitric oxide disrupter exhibits 100% sequence identity to one ofSequence ID No. 1 through Sequence ID No.
 25. 5. The method of claim 2,wherein the nitric oxide disrupter exhibits at least 80% sequenceidentity to one of Sequence ID No. 1 through Sequence ID No.
 25. 6. Themethod of claim 2, wherein the nitric oxide disrupter exhibits at least90% sequence identity to one of Sequence ID No. 1 through Sequence IDNo.
 25. 7. The method of claim 2, wherein the nitric oxide disrupterexhibits at least 95% sequence identity to one of Sequence ID No. 1through Sequence ID No.
 25. 8. The method of claim 1, wherein thesubject is characterized as exhibiting sepsis or a sepsis-relateddisorder.